Journal of Photochemistry & Photobiology, B: Biology 160 (2016) 96–101

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Journal of Photochemistry & Photobiology, B: Biology

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Highly efficient energy transfer from quantum dot to allophycocyanin inhybrid structures

A.A. Karpulevich a,e,⁎, E.G. Maksimov a, N.N. Sluchanko b, A.N. Vasiliev c,d, V.Z. Paschenko a

a Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russiab A.N.Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, 119071 Moscow, Russiac Department of Low-Temperature Physics and Superconductivity, Faculty of Physics, Lomonosov Moscow State University, 119991 Moscow, Russiad Theoretical Physics and Applied Mathematics Department, Ural Federal University, 620002 Ekaterinburg, Russiae Institute of Physical Chemistry, Hamburg University, 20146 Hamburg, Germany

⁎ Corresponding author at: AKBester Research Group, H117, 20146 Hamburg, Germany.

E-mail address: anastasi-kar@yandex.ru (A.A. Karpule

http://dx.doi.org/10.1016/j.jphotobiol.2016.03.0481011-1344/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 October 2015Received in revised form 5 March 2016Accepted 24 March 2016Available online 8 April 2016

Excitation energy transfer (EET) is observed in hybrid structures that composed of allophycocyanin and CdSe/ZnScore-shell quantum dot (QD). We demonstrate that the EET efficiency in such systems could be significantlyincreased under conditions inducingmonomerization of allophycocyanin trimers. For these purposes, the EET ef-ficiencywas estimatedunder different experimental conditions (pH, high temperature or thepresence of NaSCN)for self-assembled hybrid structures. Additionally, the hybrid structures were stabilized by covalent couplingwhich resulted in approximately 20-fold enhancement of allophycocyanin fluorescence upon excitation ofQDs. The observed effect provides new opportunities for the practical implementation of hybrid systems as fluo-rescent markers.

© 2016 Elsevier B.V. All rights reserved.

Keywords:Quantum dotsAllophycocyaninExcitation energy transferFluorescenceHybrid structures

1. Introduction

Fluorescent labeling of biological molecules is one of the commonlyused approaches in modern biotechnology. Excitation energy transfer(EET) between two types of fluorescent dyes via Fӧrster Resonance En-ergy Transfer (FRET) provides an opportunity to screen protein–proteininteractions, ligand-receptor binding and other processes [1]. However,the scope of organic fluorophores is limited due to their low stability,narrow excitation or broad emission bands [2,3].

Quantum dots (QDs) are semiconductor nanocrystals, which haveunique optical characteristics. QDs absorb light in a broad opticalrange from ultraviolet to near-infrared range, whereas their fluores-cence spectrum is narrow, symmetric and the peak position is deter-mined by the diameter of the nanocrystal [4–6]. Modern methodsallow obtaining water-soluble biocompatible QDs with high fluores-cence quantum yield, which are able to interact with different kinds ofmolecules [7,8]. Nowadays QDs arewidely used in biology andmedicineas an alternative to organic fluorescent dyes. Moreover, QDs can be en-ergetically coupledwithmolecules, increasing their effective absorptioncross-section. For instance, QDs are able to form hybrid structures withphotosynthetic light-harvesting complexes [9–12] and can even

amburgUniversity, Grindelalee

vich).

substitute native antenna complexes of photosystem II [10]. Thus, QDsmay be considered as artificial antennae.

Native light-harvesting antenna complexes are pigment-proteinstructures, the main role of which is to absorb a quantum of light andtransfer their energy to the photosynthetic reaction center. Therefore,the antennas increase an absorption capacity of the photosystem.Cyanobacteria and red algae have special light-harvesting complexescalled phycobilisomes composed of several types of water-solublephycobiliproteins, which allow them to expand the range of wave-lengths available for photosynthesis in the 530–620 nm region, inacces-sible to chlorophyll а absorption [13]. Structure of the phycobiliproteinsis conservative and relatively stable, they can easily be purified and arecharacterized by a high fluorescence quantum yield (~0.6). For thesereasons, the phycobiliproteins are widely used as model objects forprotein research [14] and as fluorescent markers in DNA microarrayand flow cytometry [15,16]. Allophycocyanin (APC) is one of thephycobiliproteins, which forms trimers in solution and emits fluores-cence at 660 nm [17]. The APC monomer consists of two subunits con-taining chromophores — the phycobilins [18–20]. Interaction of thephycobilins fromdifferentmonomerswithin the trimer causes a charac-teristic increase of absorption at 650 nm [21]. However, APC is capableof monomerization under the influence of high temperatures, low orhigh values of pH or the presence of chaotropic agents like NaSCN [21,22]. A decrease of the fluorescence quantum yield and a blue shift ofthe emission as well as disappearance of the exciton peak at 650 nmin the absorption spectrum are typical for the APC monomerization

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[20]. It is assumed that chemically cross-linked APC trimers are themostefficient for different applications such as cell sorting, high-throughputscreening and microscopy [23,24]. However, relatively low Stokes shiftand low extinction values in the blue-green region limit the range oflight sources suitable for excitation of APC.

The goal of this workwas to obtain hybrid structures of QDs and APCwith EETwhich increase the effective absorption cross-section of APC inthe blue-green region of the spectrum. We present an approach thatallowed us to improve the effectiveness of EET in self-assembling hybridstructures of QDs and APC via electrostatic interactions and especiallyby stabilizing them with a help of chemical coupling.

2. Materials and Methods

2.1. Materials

CdSe/ZnS core-shell QDs with a peak fluorescence emission at620 nm, water soluble due to the amphiphilic polymer coating and con-taining carboxyl groups on the surface, were obtained from NanotechDubna (Russia). The fluorescence quantum yield of QDs was estimatedby comparison with that of Rhodamine 6G (Sigma Aldrich) and wasequal to 0.62. Purified allophycocyanin was purchased from SigmaAldrich (USA). APC andQDsweremixed in 0.03Mpotassiumphosphatebuffer (pH 7.3) and also in 0.3 M sodium acetate buffer (pH 5.4; 4.6;3.6).

2.2. Optical Methods & Software

Absorption spectra of QDandAPСweremeasured using a L25UV/Vis/NIR spectrophotometer (Perkin Elmer, USA). To calculate APC concentra-tion we used extinction coefficient ε equal to 700,000 l·mol−1 cm−1 at652 nm [25]. For CdSe/ZnSQDs the extinction coefficientwas calculatedon the basis of the empirical dependence [26] and was equal to258,000 l·mol−1 cm−1 at 600 nm.

In order to register emission and excitation fluorescence spectra, weused Fluoromax 4 (Horiba Jobin Yvon, France). Steady-state fluores-cence emission spectra were measured in the range from 420 to750 nm with excitation at 405 nm. Fluorescence excitation spectrawere measured at 670 nm emission, whereas excitation light waschanged over the range from 400 to 650 nm. On the basis of fluores-cence excitation spectra, APC fluorescence enhancement factor A wascalculated according to the equation:

A ¼ IAD−IA−IDð Þ=IA; ð1Þ

where IAD is the APC fluorescence intensity in the presence of QD, IA isthe APC fluorescence intensity in the absence of QD, and ID is the QDfluorescence intensity in the absence of APC [27].

Time-resolved emission spectra were obtained using the time-correlated single photon counting system based on the SPC-130 mod-ule, PML-16-C detector (Becker & Hickl, Germany) and a 405 nm laserdiode (InTop, Russia) delivering 13 pJ, 26 ps (FWHM) pulses driven at10MHz. Fluorescence decay curves were approximated by a sum of ex-ponential decay functions in SPCImage software package (Becker andHickl, Germany). To compare different decay curveswe calculated aver-age fluorescence lifetime according to the following expression:

τav ¼ ∑ai � τi; ð2Þ

where τi and ai are the lifetime and the amplitude of the i-th fluores-cence decay component, respectively (the amplitude is normalized tounity: ∑ai = 1).

FRET parameters were calculated in PhotochemCAD software(LindseyLab, USA). Additionally, the isoelectric point of APC pI = 5.2was calculated by the Isoelectric Point Calculator [28].

For data evaluation, we used OriginPro 9.1 (OriginLab, USA). Graph-ical visualization of protein structure was performed in PyMol v.1.3software.

2.3. Calculation of FRET Parameters

Non-radiative energy transfer between QDs and pigment-proteincomplexes is sufficiently described by the Fӧrster theory [2]. Accordingto the Fӧrster theory, one of the main factors determining the efficiencyof EET is an overlap between donor's (QDs) fluorescence and acceptor's(APC) absorption spectrawhich is expressed by the corresponding inte-gral I:

I ¼ ʃ FD λð Þ�εα λð Þ�λ4�dλ; ð3Þ

where FD(λ) is the normalized fluorescence spectrum of the donor,εα(λ) is the molar extinction coefficient of the acceptor, λ — thewavelength. Using characteristic absorption and fluorescence spectraof the hybrid system components (Fig. 1A) and PhotochemCAD soft-ware, the integrals of the overlap were estimated to be equal to2.98 · 10−12 cm6 and 3.11 · 10−12 cm6 for the hybrid complexes ofQDs and APC in trimeric and monomeric forms, respectively. Values ofthe overlap integrals were used to calculate the Fӧrster distance R0,which characterizes the distance between donor and acceptor of theexcitation energy, when the energy migration efficiency is equal to theefficiency of intramolecular relaxation processes. The Fӧrster distanceR0 is determined by the equation:

R0 ¼ 8:8�1023�κ2�φD�I� �1=6

; ð4Þ

where φD is the fluorescence quantum yield of donor in the absence ofacceptor, κ2 — the dipole orientation factor, which can vary from 0 to 4but assumed to be equal to 2/3 for solutions and disordered systems [29].R0 was found to be equal to 103 Å and 107 Å for hybrid complexes ofQDs and APC in trimeric and monomeric forms, respectively.

EET from QD to APC is followed by the quenching of the QD fluores-cence (Fig. 1B). The EET efficiency E was quantified by a reduction ofdonor's lifetime:

E ¼ 1−τDA=τD ¼ R06= R0

6 þ r6� �

; ð5Þ

where τDA and τD are the fluorescence lifetimes of QD in the presenceand the absence of APC, respectively; r is the real characteristic distancebetween the QD and APC chromophores [4].

2.4. Chemical Coupling of the QD-APC Hybrids

Chemical coupling of APCwithQDswas conducted via randomchem-ical coupling method [30] using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide iodide (EDC) as a catalyst and N-hydroxysulfosuccinimide(sulfo-NHS) as a stabilizer of the EDC intermediate (all reagents werepurchased from Sigma Aldrich). Conjugation was carried out in 0.03 Msodium phosphate buffer (pH 7.3) at a QD to APC molar ratio of 1:1.The final EDC and sulfo-NHS concentrations were 400 μM and 500 μM,correspondingly. QD solution was mixed with EDC and then sulfo-NHSwas added to the reaction tube. After 5min of incubationwith gen-tle shaking, the APC was added. The APC monomerization was inducedprior to the coupling either by incubating the sample for several hoursat 50 °C, or by addition of NaSCN up to the final concentration of0.25 M [20]. After the coupling the sample was centrifuged at12,000 rpm for 30min at 20 °C to remove uncoupled APC. The superna-tant was removed and the sediment was dissolved in a sodium phos-phate buffer. The series of controls were carried out to estimate theeffect of crosslinking chemicals on individual properties of QDs andAPC. We did not observe significant influence of these chemicals onthe QD and APC optical properties.

Fig. 1. (A) Normalized absorption (abs) and fluorescent (fl) spectra of CdSe/ZnS QD and APC; green area indicates the overlap of APC absorption and QD fluorescence. (B) Fluorescencedecay curves of QD at 620 nm in the absence and in the presence of APC (self-assembled hybrid structures in potassium phosphate buffer, pH 7.3, room temperature). (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Pure APC is characterized by a low sedimentation factor and normal-ly does not precipitate under used conditions. We also did not observeany significant sedimentations of APC in controls containingcrosslinking chemicals in the absence of QDs. Therefore, the presenceof typical for APC absorption and emission peaks in the resuspendedsediment was considered as a criterion of successful conjugation.

Obtained hybrid structures remained stable during several weeks.

3. Results and Discussion

In order to estimate the efficiency of EET at different donor/acceptorratios we performed titration of QDs (56 nM) by gradually increasingthe concentration of APC (Fig. 2).

Hybrid structures in solution can be formed as a result of (1) electro-static interactions between the negatively charged carboxyl groups onthe surface of QD and the positively charged amino acid residues ofthe protein; or (2) due to the random collisions of uniformly distributedQDs and APC in solution. However, due to nM concentrations of compo-nents the contribution of (2) is not significant and therefore the electro-static interactions (1) should have a major effect. At pH 7.3 both COOH-coated QDs and the surface of APC have a negative net charge (isoelec-tric point pI of APC is equal to 5.2). This may cause the electrostaticrepulsion between QDs and APC at neutral pH resulting in low EET

Fig. 2. The EET efficiency as a function of the APC-to-QDmolar ratio at different pH values.The EET efficiency was calculated as a relative reduction of QDs fluorescence lifetime (seeMethods for the detailed explanation). Each point on the graph is an average of threeexperiments.

efficiency. To test this hypothesis, we analyzed the EET efficiency atdifferent pH values (Fig. 2).

We found that the reduction of pH caused significant increase of theEET efficiency. It is clear from Fig. 2 that at pH 7.3 and 5.4 the EET effi-ciency E is slightly above 30%, whereas at pH 4.6, and especially atpH 3.6, E values raise up to 90%. Note that at low pH the EET efficiencyreaches a plateau at APC to QD concentration ratio above 0.4. As it wasmentioned, in solution APC is capable of monomerization under certainexperimental conditions. One can easily detect it due to characteristicchanges in the absorption spectrum. Indeed, at pH values lower thanthe isoelectric point of APC (pI = 5.2) we observed the reduction ofthe 650 nm band in steady-state absorption spectra (Fig. 3), corre-sponding to APC monomerization.

To check that the EET increase is caused by the interactions of QDand APC in the monomeric state, we applied two other well-knownmethods of the APC monomerization — relatively high temperature(up to 50 °С) and treatment by chaotropic agent NaSCN [18,20,21].We performed titration of APC by QDs at neutral pH under either ofthe mentioned conditions. In these experiments we observed thesame rise of the EET efficiency up to 90% as it was at pH 3.6 (seeFig. 2) and the maximum efficiency was reached at APC to QD concen-tration ratio equal to 1:3, in other words, 1 APC monomer to 1 QD. Itshould be noted that NaSCN noticeably affected optical properties ofQDs and led to a significant reduction of fluorescence lifetime of QDseven in the absence of APC (data not shown). This effect is known ascyanide-susceptibility of CdSe QDs [31] and should be investigatedfurther.

The main FRET parameters of the hybrid structures containing APCin two different assembly states are summarized in Table 1.

According to our calculations (Table 1), the distance separating QDand the chromophore in the APC monomer is approximately 1.68times shorter than that of APC in the trimeric state. At the same time,the Fӧrster distance is similar for both types of hybrid structures, indi-cating that fluorescence of the donor and absorption of the acceptorhave significant overlap in both cases. According to the Fӧrster theory,the EET efficiency E is inversely proportional to the distance betweendonor (QD) and acceptor of energy (APC chromophore) in a power ofsix (see Eq. (5)). Thus, it is possible to explain the observed increase ofthe EET efficiency by the reduction of the distance between QD andchromophores of APC in the monomeric state. Indeed, in the trimericstate of APC chromophores are covered by the apoprotein and theneighboring subunits, whereas in the monomeric APC chromophoresare in the immediate vicinity to the surface. Simultaneously, the dipoleorientation factor κ2 also contributes to the EET efficiency (see Eqs. (4),(5)), and therefore the stabilization of the hybrid structuresmay furthersubstantially improve the EET. Previously, we visualized equipotentialsurfaces of the APCmonomers at pH equal to 3 and 7 using a homemadeplugin for PyMol [32]. Surprisingly, some parts of the protein near the

Fig. 3. Schematic of the structural and optical characteristics of the APC trimer (red) and monomer form (black). Fluorescence (fl) and absorption (abs) spectra of each APC state werenormalized for better presentation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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regions containing chromophores remain negatively charged even atlow pH. Despite this fact, the EET efficiency in the hybrid structures ob-tained due to thepH-inducedmonomerization of APC is as high as in thecase of temperature- or NaSCN-induced APC monomerization. This factprobably indicates that (1) electrostatic interactions do not provide spe-cific configuration of the hybrid structure and (2) the number of suchconfigurations is considerably lower in the trimeric state of APC due tothe geometry of the trimer and its internal protein–protein interactions.Thus, we assume that steric accessibility of APC chromophores plays anessential role in the EET efficiency in QD-APC hybrid structures.

Next, to estimate how the stabilization of the donor and acceptorconfigurations affects EET, we obtained chemically coupled hybridstructures of QD and APC (see Section 2.4). Selected method of randomchemical coupling allows the conjugation of the COOH-groups presenton the QD surface with the NH2-groups of Lys' residues in APC. Basedon 1ALL structure [17], 13 Lys could be found on the surface of APC, pro-viding a good opportunity for the chemical coupling.

Our approach allowed us to separate uncoupled APC, however, afterall procedures, we obtained amixture of QDs and QDs linked to APC. Es-timation of QD fluorescence decay component yields revealed that onlyabout 10% of QDs are quenched due to the coupling to APC. We believethat additional purification with a help of e.g. affinity chromatographyand a His-tagged APC will allow obtaining more pure and near-stoichiometric QD-APC hybrid structures in future studies.

By registering the APC emission upon excitation of QDs at differentwavelengths, we can analyze the contribution of QDs energeticallycoupled to APC. For this purpose, fluorescence excitation spectra arevery useful. Fig. 4 shows fluorescence excitation spectra of the hybridstructure obtained after the temperature-induced APCmonomerizationand control spectra of individual QD and APC, normalized to their ab-sorption. The considerable contribution of APC absorption in 550–660 nm region was expected (it should be noted that the shape of thespectrum and the presence of characteristic 650 nm band reflect thepresence of APC trimers, indicating that after elimination of themonomerization factor the APC monomer coupled to QD can assemble

Table 1FRET parameters of self-assembledhybrid systems ofQDandAPC in the trimeric or themonomepH= 7.3); The “QD-APCmonomer” represents average parameters for hybrids obtained upon pH

Type of the hybrid system The EET efficiencyE, %

QD-APCtrimer 30 ± 5QD-APCmonomer 90 ± 8

with other monomers). We found that APC fluorescence in the hybridsystems obtained after monomerization under excitation in a range of400–550 nm is significantly higher than the sum of free APC and QDemissions at the corresponding concentrations (Fig. 4). This fact indi-cates that the energy absorbed by QD transfers to the APC. Thus, QD in-creases the effective absorption cross-section of APC over the 400–550 nm region. To quantify this effect and to compare different typesof chemically coupled hybrid systems we calculated the APC fluores-cence enhancement factor A (see Fig. 5).

It is seen from the Fig. 5, that monomerization of APC prior to chem-ical coupling of the hybrid systems can significantly increase the EETcoupling and effective absorption cross-section of APC in the blue-green region of the spectrum. Earlier it was mentioned that NaSCNcauses the quenching of QDs, but even this method of the APCmonomerization results in more efficient involvement of QD in theEET as compared to the QD self-assembling with the APC trimers. Nev-ertheless, the temperature-induced monomerization was found to bethe most effective way of increasing the APC absorption cross-sectionby EET from the chemically coupled QD. In these experiments the APCfluorescence intensity in the covalently coupled hybrid system was al-most twenty times higher than of the APC trimers.

4. Conclusions

It was shown that QD and APC are capable of forming self-assemblinghybrid structures, presumably, due to the electrostatic inter-actions. While the excitation energy transfer (EET) coupling of QDs andAPC in the trimeric form is rather ineffective, monomerization of APC al-lows improving the efficiency of the EET. Monomerization of APC in-duced by low pH, relatively high temperature or NaSCN led to anincrease of the EET efficiency from 30% to 90%. We suggest that themain reason of ineffective energetic coupling in the self-assembled hy-brid structures of QDs and the APC trimers is the limited steric accessi-bility of the APC chromophores due to protein–protein interactionswithin the trimer. Additionally, the covalently coupled hybrid structures

ric state. TheQD-APCtrimer hybridswere obtained atneutral conditions (room temperature,-, temperature- or NaSCN-induced monomerization of APC.

The Fӧrster distanceR0, Å

The distance r between QD and APC, Å

107 123105 73

Fig. 4. Fluorescence excitation spectrum of the covalently bound QD-APC hybridstructures (black) obtained after the temperature-induced monomerization andexcitation spectra of control samples, containing similar concentrations of QD (red) andAPC (blue) in the trimeric state. Fluorescence intensity was registered at 680 nm,whereas excitation wavelength ranged from 390 to 660 nm. The difference curvecorresponds to IAD −IA − ID (see Eq. (1)). (For interpretation of the references to colorin this figure legend, the reader is referred to the web version of this article.)

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of QD and APC were obtained via a random chemical coupling method.Itwas found that QDs can increase the effective absorption cross-sectionof APC in the blue-green region of the spectrum due to the more effi-cient EET. Monomerization of APC prior to the chemical coupling ofAPC and QD allowed us to further increase the APC fluorescence withthe total increase of up to 20 times.

Thus, the results of this research demonstrate the possibility to syn-thesize stable hybrid structures consisting ofQDandAPC inwhich effec-tive non-radiative energy transfer occurs. Allophycocyanin has practicalimplementation as afluorescentmarker inDNAmicroarray andflow cy-tometry, but transition into monomeric state is often considered as adisadvantage for its fluorescent properties. Our results show that thisdisadvantage can be turned into an advantage with the help of modernnanotechnology.

Fig. 5. The APC fluorescence enhancement factor A calculated as a function of theexcitation light wavelength (see Eq. (1)). In the chemically coupled QD-APC hybridsystem A was obtained upon the temperature-induced (blue) and NaSCN-induced(green) monomerization of APC. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

Author Contributions

M.E.G. and K.A.A. designed and performed the optical experiments,S.N.N. and K.A.A. designed and performed the synthesis and basic char-acterization of hybrid structures, and P.V.Z. andV.A.N. contributed to thedesign of the experiments, analysis and discussion of the results. K.A.A.wrote the manuscript with an input from all authors.

Acknowledgments

The authors are grateful to G.S. Budylin, A.F. Prohorova and Dr. E.P.Lukashev for their help and valuable advices. V.Z. Paschenko and E.G.Maksimov thank the Russian Foundation for Basic Research (projectsNo. 14-04-01536A, 15-04-01930A, 15-29-01167) for partial support ofthis work. E.G. Maksimov thanks the Russian Ministry of Educationand Science (project MK-5949.2015.4) for partial support of this work.This work was supported, in part by a Dynasty Foundation Fellowshipto E. Maksimov. A.N. Vasiliev acknowledges the support of RussianScientific Foundation (project No.14-50-00029). The reported studywas funded by RFBR andMoscow city Government according to the re-search project No. 15-34-70007 «mol_а_mos». The work was supportedby Act 211 Government of the Russian Federation, contract No.02.A03.21.0006.

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